Purification of chlortetracycline



United States Patent @fifice greases Patented Sept. 1%, 195%PURIFICATIUN Gi CHLQRTETRACYCLINE Robert Winterbottom, Pearl River,Peter Eiclller, New

City, N. Y., and Charies Pidaclrs, Montvaie, N. J., assignors toAmerican Cyanamid Company, New York, N. Y., a corporation of Maine NoDrawing. Application March 3, 1954, Serial No. 413,946

Claims. (Cl. 260-559) This invention relates to a process for thepurification of chlortetracycline, and obtaining chlortetracycline asneutral chlortetracycline or its alkali salts, and has for its generalobject an improvement in methods of obtaining chlortetracycline.

The novel method is particularly useful in converting various forms ofchlortetracycline to comparatively pure neutral chlortetracycline or itsalkaline salts. The method is also useful in improving the purity ofcrude chlortetracycline. As a result, the chlortetracycline has betterappearances, and is more acceptable from'a therapeutic point of view.The more highly purified products are more desirable for therapeuticpurposesbecause extraneous materials which may be present in some casescause undesired physiological side reactions.

This application is a continuationinpart of our copending applicationSerial Number 142,074, filed February 2, 1950, now Patent Number2,671,806, March 9, 1953 entitled Purification of Chlortetracycline.

Chlortetracycline is produced and sold under the commercial designationAureomycin, which, in certain countries, is a trademark of the AmericanCyanamid Company. Chlortetracycline has been found to be a naphthacenederivative and while having a structure which strongly suggeststautomeric forms, is believed to exist with the following probablestructure:

OHO

O ONHz H H N H CH3 OH Ca \CH;

A numbering system is shown. The name under which the compound isindexed in Chemical Abstracts is 7-chloro-4-dimethylamino-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy-6-methyl-1,1 l-dioxo-Lnaphthacenecarboxamide[Chemical Abstracts, vol. 46, page 13363 (1952)]. Other numberingsystems have been used in other U. S. patents, such as 2,658,077,andelsewhere. The accepted generic term for the compound ischlortetracycline. Chlortetracycline is produced by-growing Streptomycesaureofaciens asdescribed and claimed in Patent 2,482,055, September 13,1949 to B. M. Duggar entitled Aureomycin and Preparation of Same. As soproduced, certain contaminants and impurities are present, which can becalled fermentation mash impurities. The present process removes suchfermentation mash irnpurities to give a purer chlortetracycline.

Chlortetracycline is a very unusual antibiotic in that it is amphoteric,having both acidic and basic groups in its molecule, and accordingly attimes acts astan acid, and at times actsas a base. Accordingly, methodspreviously used to purify acid or basicantibiotics are not generallyuseful for chlortetracycline. Additionally, it has certain very specialproperties because there is a tendency to- 2 Wards partialpolymerization or rearrangements which inactivate the chlortetracyclineunless it is treated particularly gently.

Because of the unstable and amphoteric properties of chlortetracycline,it has been necessary to use an entirely different concept ofpurification than has ever been previously known in the study ofantibiotics. Additionally, certain of the impurities have a tendency toremain with the chlortetracycline when using ordinary methods ofpurification; but by purification in accordance with the procedures ofthis invention, it is possible to obtain high yields of therapeuticallyacceptable material; obtaining the chlortetracycline as the neutralchlortetracycline, or as asalt with an alkali base, for example, asodium salt,

each of which have particular advantages for therapeutic .use, dependingupon the condition to be treated, the mode of administration, etc.

An object of this invention is to prepare the chlortetracycline in atherapeutically etfective form with the largest practical yield, in ascheap and efiicient a manner as possible. Furthermore, it is an objectof this invention to prepare the alkali salts, or the neutralchlortetracycline which may be desired for a particular medicaltreatment, with the least expensive and troublesome modification of thepurification procedure. By following the purification procedures of thisinvention, it is possible for the preliminary steps, solution,filtration, and so on, to be carried out under identical conditions,independent of the desired final product form, and the conversion to thedesired final form be accomplished shortly before the final isolation ofthe purified material, so that a major process modification is notnecessary to obtain the particular form of chlortetracycline which maybe then desired.

Neutral chlortetracycline and the sodium salt are also highlysatisfactory for therapeutic use and may be obtained in crystalline formof high purity by the process of the present invention. Other salts ofchlortetracycline with bases such as the potassium salt, the ammoniumsalt, the lithium salt, the cesium salt, the rubidium salt, and

the monoamine salts are all easily prepared through the use of ourprocess, and Where conditions justify, or under circumstances where themedical profession prefers them, such salts are easily made by followingthe procedures taught by this invention, and are included within its itis more convenient to use a nitrogeneous base for the pH control, or itis easier to prevent local over alkalinization and the residual base ismore easily removed from the desired material.

A great many of the impurities naturally present at the time of theoriginal formation of chlortetracycline will be found to becomparatively insoluble in this alkalinized solution and may be removedby filtration; and many materials, particularly the color bodiespresent, may be adsorbed upon a charcoal or diatomaceous earth which maybe present. An activated charcoal, such as Darco G 60, is found to bevery' satisfactory. From this alkalinized solution, thechlortetracycline may be recovered in the desired form by altering thesystem characteristics.

To obtain the neutral chlortetracycline, the preferred range is betweenabout a pH of about 4 and a pH of about 7.5 with a pH of about 6 givingparticularly effective results. To obtain the chlortetracycline as analkali salt such as, the sodium, potassium, lithium, cesium, rubidium,ammonium, or mono-amine salt, the chlortetracycline may be recoveredfrom the solution as formed within the range of about 8 to 10. Thechlortetracycline in the desired form is caused to become insoluble bythe shift in the pH, combined with a change in the solvent system andthe temperature. Below a pH of about 4, at least some of an acid salt ofchlortetracycline is also obtained. In the range of about 7.5 to 8 someneutral chlortetracycline is obtained with the alkali salt. These rangesmay be used if mixtures are desired, or the mixtures may be separated bypartial crystallization.

Among the polar solvents which are suitable are such solvents as methylalcohol, ethyl alcohol, higher alcohols, Z-methoxyethanol, 2-ethoxyethanol, (alkoxyalkanols) ethylenechlorhydrin, dioxane, carbitol, ethyleneglycol, benzyl alcohol, nitromethane, acetonitrile,propylenechlorhydrin, di-acetone alcohol, acetophenone, Z-methoxyethylacetate, and mixtures of two or more of these solvents. The loweralkoxyalkanols and lower alcohols are generally preferred because thechlortetracycline has better solubility characteristics herein. Methylalcohol and Z-methoxyethanol and 2-ethoxyethanol are particularlyeffective as they are readily obtainable commercially, and arecomparatively inexpensive. If for any reasons these lower members areunavailable, or for other commercial reasons others are desirable, thehigher members may be very effectively used. Ethyl alcohol and methylcarbitol are among the solvents which are very effective, althoughsomewhat more expensive. A little water in these solvents, oftenincreases the solubility of the chlortetracycline and permits the usageof smaller solvent volumes. Addition of larger amounts of water up tothe limits of miscibility aids in the crystallization of the neutralchlortetracycline or its alkali metal salts. Excellent yields of neutralchlortetracycline are'obtainable by dissolving the crudechlortetracycline in anhydrous methanol or ethylenechlorhydrin followedby the addition of water. Inactivation of the chlortetracycline isusually less with less water present, particularly at the highertemperatures or at the more alkaline ranges.

Solubilities are among the most obscure of all known properties of amaterial. It is frequently found that, for no apparent reason, slightchanges in the structure of the product or of the solvent will causelarge changes in the relative solubilities. It is particularlysurprising that, particularly with the mono-amines, and othernitrogenous bases such as ammonia, to neutralize the acid group on thechlortetracycline and the acidic radical of any salt which may bepresent, the solubility of the chlortetracycline in hydroxylated andethereal organic solvents is increased to such a remarkable extent.However, it is not a sharp phenomenon, and the amounts of ammonia oramines to solubilize, and the exact pHs, vary to some extent with theconcentration and solvent which is being used.

The neutral chlortetracycline may be used as the starting materialrather than an acid salt, in which case a smaller quantity of the baseis required for effective solubilization. The quantity of base to beused may either be on a calculated basis from the purity and quantity ofchlortetracycline which is being used, or it may be by an actualmeasurement of the pH involved. For the determination of pH ofnon-aqueous solutions, the term pH in and of itself, to a large extentloses its significance; but as a practical operating value, it is foundthat by diluting an equal volume of a solvent with water and thenmeasuring the pH of the aqueous layer, if immiscible, or the mixture, ifmiscible, with a standard glass electrode, there is obtained a readingof pH, or acidity and basicity, which is in effect a very useful figure,even though it may not exactly come within the scope of the classicdefinition of that which is meant by pH as originally defined. For thepurposes of this invention, where a non-aqueous solvent is being used,the figure obtained in this manner is referred to as the pH of thesolution.

The amines are more desirable than the metallic alkalis if neutralchlortetracycline is to be recovered. It is desirable that a non-toxicamine be used, and preferably one of low molecular weight and low costso that the expense involved will be a minimum, the quantity will be aminimum, and the necessity for a thorough removal of the amine will beat a minimum. Of course, by suitable precautions any of these factorsmay be neglected if considered desirable for a specific set ofconditions. Ammonia is, of course, considered the first member of thefamily of amines, or nitrogenous bases, in which none of the hydrogensare substituted by organic radicals; and because of this fact a lesserquantity may be used for neutralization. Additionally, ammonia iscomparatively non-toxic, and if ammonia is permitted to remain in theform of an ammonium halide, it is a harmless diluent.

Triethylamine is particularly useful because it is" readily obtainablecommercially, it is of a comparatively low molecular weight, and is ingeneral highly useful as it causes rapid solubilization and is easilymanipulated. Other amines, such as diethylamine, tri-n-butylamine,cyclohexylamine, morpholine, di-n-propylamine, betaphenylethylamine,ethanolamine, isoamylamine, and ethyl morpholine, dimethylbenzylamine,Z-aminopyridine, isobutylamine, dicyclohexylamine, diethanolamine,triethanolamine, beta-di-ethylaminoethanol, and trimethylamine, andmixtures of any of the suitable bases give satisfactory results,although their recovery cost or scarcity renders them less desirablefrom a commercial standpoint.

In general, the amines which have an ionization constant of 10 orstronger (as bases) are satisfactory. Those with an ionization constantof 10" or stronger, are better because they operate more rapidly andinsur greater solubility.

It is desirable that the solution be fairly close to neutral becausechlortetracycline is subject to decomposition if it is permitted tobecome too basic, particularly in the presence of water, or when hot.With ammonia, or triethylamine, etc., the natural weaknesses of the baseserves to prevent the solution from becoming too basic, even locally,during the preliminary mixing. Even with these, losses are reduced ifthe mixture is kept cool, e. g. 5 C. while on the alkaline side duringprocessing.

The chlortetracycline, which may be either as a free base or any of itsacid salts is normally most conveniently suspended in the solvent as aslurry, and thereto added the base with stirring, whereby thechlortetracycline is converted to its soluble form on the alkaline side,although of course, other orders of addition or mixing may be used.

The insoluble impurities which are usually present, unless unusualprecautions had been previously taken to obtain their removal, may thenbe removed by filtration or other means. Color impurities which arepresent are removable with a decolorizing carbon or filteraid, which maybe added at this time.

After the removal of the impurities, the chlortetracycline is separatedfrom the clarified solution as the desired form. This may also beconveniently done at room temperature, although if the material is toremain for any length of time, it is desirable that it be fairly cool toprevent the chlortetracycline from decomposing, particularly if thematerial is in the more alkaline of the above-mentioned range. From thissolution of the chlortetracycline, the material desired may be recoveredin three forms, the acid form as specifically claimed in our PatentNumber 2,671,806, the neutral form, and as an alkali salt.

For the recovery of neutral chlortetracycline, it is possible to addsufiicient acid to lower the pH to within the neighborhood of 4 to 7.5preferably about 6, at which point the neutral chlortetracycline isformed in the solution and may be separated therefrom. Whereas any ofthe solvents above mentioned may be used for the recovery of thisneutral chlortetracyeline, methanol and the cellosolves give solutionswhich, on partial neutralization so as to form the neutralchlortetracycline, causes the neutral chlortetracycline to come out inaform which is more readily separated from the solvent. With certain ofthe other solvents, there is more of a tendency for the formation of agelatinous, or diIficultly-filterable, precipitate.

For the recovery of the alkali salts, it is possible to, with the use oforganic solvents, add water thereto and cause the chlortetraeycline inthe form of its alkali salt to become less soluble and precipitate out.For commercial yields, however, it is normally preferred to use anorganic solvent, such as methanol, for the recovery of the alkali salt,rather than water alone. This procedure is particularly effective informing the sodium and potassium salts.

The separation of the desired form .of chlortetracycline may be fairlyslow, so that complete solution of the chlortetracycline occurs, andwith standing, particularly with cooling, separation of the desired formmay occur without the necessity for diluting out the solvent.

Similarly, a transformation of form may be accomplished in too small avolume of solvent for complete solution.

After the separation of the chlortetracycline in the form of the desiredsalt, it is convenient that it be washed to remove the solvent, and anyimpurities, in accordance with the usual crystallization procedures. Thesolvent need not be the same as that in which the chlortetracycline wasoriginally dissolved; but to simplify the problems of solvent recovery,it is most convenient to use the same solvent. It is convenient to Washthe crystals as recovered, first with the solvent with which they wereoriginally formed; then with a small quantity of water; and then withalcohol, although the order is not important. Water tends to remove anysalts which may be present, such as those of ammonia or the amines whichmay have been formed simultaneously with the desired salt ofchlortetracycline. The material is then dried for use.

To better exemplify certain specific modifications of our invention,specific examples are herewith set forth.

EXAMPLE .1

Sodium chlortetr acycline 100 grams of a crude chlortetracyclinehydrochloride was slurried in 500 cc. of 2-ethoxyethanol. Thereto wasadded 35.8 cc. of 10.8 normal sodium hydroxide, :and the mixture stirreduntil solution resulted. Any insoluble impurities were remoyedbyfiltration and to the clear filtrate was added 100 cc. of distilled.water. The mixture was allowed to stand, with stirring, for /2 hour atroom temperature, and then placed in a chill room overnight. Anorange-yellow precipitate formed, which was filtered, washed twice witha 6:1 2-ethoxyethanol-water wash mixture, and once with anhydrousethanol. The crystals thus formed were dried over phosphorus pentoxide,and thereby was obtained a yield of 621 grams. of orange-yellow crystalsof the sodium salt ofchlortetracycline, assaying 890 micrograms permilligram, according to the. standard assay. The sodium salt ishygroscopic and must be kept dry to prevent its picking up an unduequantity of water.

EXAMPLE" 2 Chlorletracycline 60 grams of a comparatively dry sodiumchlortetracycline, assaying 890 micrograms per milligram, were slurriedin 300 cc. of Z-ethoxyethanol. To the slurry was added 18.1 cc. of 6.8normal hydrochloric acid, thereby resulting in a solution, which whendiluted with 6 ante'qual quantityof water, gave a pH of6175. To the 2e'thoxyethanol solution was added 600 cc. of distilled water; withstirring, over a period of 1 hour. The pH was found to be 7.7 and wasadjusted to 7' with 3.0 cc. of the hydrochloric acid. The mixture was.chilled for 2 hours, and the crystals formed removed by filtrationandwashed three times with water. The crystals were dried at roomtemperature over phosphorus pentoxide for 12 hours, and thereby wasobtained a yield of 39.1 grams of neutral chlortetracycline, assaying1100 micrograms per milligram. This is a recovery of 81% of thechlortetracyeline activity.

EXAMPLE 3 Sodium chlorletr'acycline A suspension was prepared of 30grams of crude chlortetracycline hydrochloride and cc. of2-ethoxyethanol. To this suspension was added suificient of 10 normalsolution of sodium hydroxide to raise the to 8.5. The mixture wasstirred rapidly, to prevent local over-alkalinization, and care was usedto see to it that the entire amount of caustic was added within a shortperiod. The solution was comparatively clear. Thereto was added 1 gramof decolorizing carbon, the mixture stirred, allowed to settle, and thenfiltered. Much of the color and many of the impurities were therebyremoved.

T o 50 cc. of this clear filtrate was added an equal volume of water,the mixture cooled with stirring, and allowed to stand overnight in achilled chamber; and then the sodium salt of chlortetracycline which wasthere byprecipitated separated by filtration. The sodium salt was washedonce with ethyl alcohol, then with ether, and dried. A pale, dry, yellowmaterial was obtained.

EXAMPLE 4 Chlortetracyline To a second 50 cc. portion of the filtrate,obtained in the preceding example, was added sufiicient hydrochloricacid to lower the pH to 6.0. The mixture was'stirred, and then there wasadded thereto 50 cc. of distilled water, and the mixture allowed tochill overnight in a refrigerator at 4 C. forming crystals. The thuscrystallized chlortetracycline (neutral) was separated by filtration,washed once with water, once with ethyl alcohol, and then permitted todry. A pale, yellowish, crystalline, neutral chlortetracycline wasthereby obtained.

EXAMPLE 5 Chlortetracycline 50 grams of chlortetracycline, neutral, wassuspended in 250 cc. of 2-..thoxyethanol. Sutficient 10 normal sodiumhydroxide was added to raise the pH to 7.5. The mixture was warmed toabout 40 C. to hasten solution. The solution was filtered from anyinsoluble impurities, and to the filtrate was added 250 cc. of water.Chlortetracycline (neutral) crystallized out rapidly. Thelong yellowneedles of neutral chlortetracycline were removed by filtration, washedwith 1:1 Z-ethoxyethanol-water solution, then ethyl alcohol, then ether,and dried. A total of 35.3 grams of chlortetracyclinewere recovered asthe neutral chlortetracycline with a potency of 950 micrograms permilligram".

EXAMPLE 6 Chlortetracycline To 25 grams of chlortetracyclinehydrochloride was added 200 milliliters of methanol and 6.4 millilitersof triethylamine, and the resulting mixture stirred to give a solutionat a pH of 5.03. The insolubles were filtered off, and the filter washedwith 25 milliliters of fresh methanol. To the solution was added 33milliliters of water over a 15 minute period with constant stirring, andchlortetracycline, neutral, was thereby precipitated. Stirring wascontinued for an additional hour, and the material was kept at 4 C.overnight. The chlortetracycline (neutral) was filtered, washed twicewith 25 milliliters of 85% methanol and dried in vacuo. A 90% recoveryof chlortetracycline Was obtained, calculated on the purity involved.The resulting material analyzed 1030 micrograms per milligram, using astarting material with an analysis of 850 micrograms per milligram.

EXAMPLE 7 Chlortetracycline To 275 grams of chlortetracyclinehydrochloride was added 2200 milliliters of anhydrous methanol, and 72milliliters of triethylamine. The mixture was thoroughly stirred andfound to have a pH of 5.3. The insolubles were removed by filtration andwashed with 180 milliliters of methanol, the wash being added to thefiltrate. The final volume of solution was 2540 milliliters. To this wasadded 20% by volume of distilled water, the mixture stirred, permittedto stand for 16 hours at 4 C., and the chlortetracycline therebyprecipitated was filtered, washed twice with 250 milliliters of 80%methanol and dried in vacuo. There was obtained a unit yield of 95% ofthe total chlortetracycline originally present, and the final materialwas found to analyze 970 micrograms per milligram.

EXAMPLE 8 Ch lortetracycline 25 grams of crude chlortetracyclinehydrochloride were slurried in 200 milliliters of methanol. The pH wasadjusted to 5.71 with 6.0 milliliters of ethyl morpholine. The solutionwas filtered, and the filter pad washed with 25 milliliters of methanol.chlortetracycline was precipitated by the addition of 30 milliliters ofwater with stirring. After standing for 16 hours, the chlortetracyclinewas filtered, washed twice with 20 milliliters of 80% methanol and driedin vacuo. There was obtained a yield of 87% of material analyzing 1010micrograms per milligram of chlortetracycline. The Weight of the productwas 19.8 grams.

EXAMPLE 9 Chlortetracycline The above experiment was repeated exceptthat ethanolamine was used as the nitrogenous base, and there wasobtained 18.7 grams of chlortetracycline analyzing 990 micrograms permilligram.

EXAMPLE 10 Chlortetracycline The above experiment was repeated usingbeta-phenylethylamine as the nitrogenous base, and there was obtained ayield of 19.6 grams of chlortetracycline with purity of 905 microgramsper milligram.

EXAMPLE 11 chlortetracycline The above experiment was repeated usingtriethylamine as the nitrogenous base, and there was obtained a yield of19.4 grams of chlortetracycline analyzing 1000 micrograms per milligram.

' 8 EXAMPLE 12 Potassium chlortetracycline To a slurry of 10 grams ofchlortetracycline hydrochloride in milliliters of methanol and 5milliliters of water, 2 equivalents of 10 N aqueous potassium hydroxidewas added. On slight warming a solution formed which was filtered toremove insolubles. After cooling in an ice bath for 15 minutes theresultant heavy precipitate was filtered off and washed with coldmethanol and dried. A yield of 6.6 g. of the potassium salt ofchlortetracycline was obtained on drying. This substance assayed 800micrograms per milligram and was soluble to the extent of 10% in water.

EXAMPLE l3 Ammonium chlortetracycline Four equivalents of 28% aqueousammonia was added to a slurry of 5.0 grams of chlortetracyclinehydrochloride in 30 milliliters of methanol. Although solution of thesolids did not occur their crystalline structure was visibly altered.The slurry was stirred for three hours at 4 C. and filtered. Theprecipitate was washed with methanol and dried to yield 2.4 grams of theammonium salt of chlortetracycline assaying 1030 micrograms permilligram. This material is water soluble unlike the neutral form ofchlortetracycline.

Many obvious modifications, involving slight changes in temperatures,pressures, concentrates, etc., as well as minor mechanical modificationssuch as centrifugations or decantations instead of filtrations, etc.,will suggest themselves to those skilled in the art.

We claim:

1. A process for the preparation of neutral chlortetracycline whichcomprises dissolving crude chlortetracycline hydrochloride in a solventselected from the group consisting of lower-alkanols andlower-alkoXy-loWer-alkanols by the addition of an amine having anionization constant of 10- or greater at a pH in the range of 4-7.5,filtering to remove the insoluble impurities, and adding water to thesolution whereupin purified neutral chlortetracycline precipitates.

2. A process for the preparation of neutral chlortetracycline whichcomprises dissolving crude chlortetracycline hydrochloride in a solventselected from the group consisting of lower-alkanols andlower-alkoXy-lower-alkanols by the addition of a compound selected fromthe group consisting of triethylamine, ethyl morpholine, ethanolamine,and ,B-phenylethylamine at a pH in the range of 4-7.5, filtering toremove the insoluble impurities, and adding water to the solutionwhereupon purified neutral chlortetracycline precipitates.

3. A process as in claim 2 in which the amine is triethylamine.

4. A process as in claim 3 in which the solvent is methanol.

5. A process as in claim 3 in which the solvent is 2- ethoxyethanol.

References Cited in the file of this patent UNITED STATES PATENTS2,482,055 Duggar Sept. 13, 1949 2,516,080 Sobin et al July 18, 19502,640,842 Weidenheimer et al June 2, 1953 2,671,806 Winterbottom et a1.Mar. 9, 1954 OTHER REFERENCES Harned et al.: Annals, N. Y. Acad. Sci.vol. 51, art. 2, Nov. 30, 1948, p. 183.

1. A PROCESS FOR THE PREPARATION OF NEUTRAL CHLORTETRACYCLINE WHICHCOMPRISES DISSOLVING CRUDE CHLORTETRACYCLINE HYDROCHLORIDE IN A SOLVENTSELECTED FROM THE GROUP CONSISTING OT LOWER-ALKANOLS ANDLOWER-ALKOXY-LOWER-AL KANOLS BY THE ADDITION OF AN AMINE HAVING ANIONIZATION CONSTANT OF 10-7 OR GREATER AT A PH IN THE RANGE OF 4-7.5,FILTERING TO REMOVE THE INSOLUBLE IMPURITIES, AND ADDING WATER TO THESOLUTION WHEREUPIN PURIFIED NEUTRAL CHLORTETRACYCLINE PRECIPITATES.